Heart malformations are frequently occurring, clinically significant birth defects that are environmentally-linked and involve maldevelopment of "endocardial cushion tissue (ECT)", the primordium of valves and internal septa. We have shown that ECT morphogenesis is a cascading sequence of: induction, epithelial/mesenchymal transition, cell migration, histodifferentiation and cell-cell adhesions (fusions). "Induced" endocardium of specific heart regions transforms into cushion mesenchyme; migration and interstitial growth of the latter form opposing ECT pads which project into the lumen of the atrioventricular or bulbus cordis and ultimately "fuse" creating internal partitions. Based on spatial and temporal correlations (established during the tenure of this grant) between specific extracellular macromolecules (ECM) and the basic, sequential steps in ECT morphogenesis we propose that (1) endocardium is post-translationally programmed to form ECT by contact with endoderm or endodermally derived ECM; (2) endocardium is "induced" to initiate the complex, multifaceted process of transition into mesenchyme by either one or more of the 35 glycoproteins or by a large proteoglycan (PG) of chondroitin sulfate secreted by the myocardium. Those factors which serve to complete this transition - i.e., pericellular heparan sulfate PG and/or fibronectin - also mediate sustained mesenchyme migration upon native collagen and facilitated ECT pad fusion. Lastly, a tissue specific PG secreted by migrating mesenchyme as "footprints" upon their collagenous substrates serves to initiate histodifferentiation into valvular tissue. These hypotheses will be tested using ECM isolated (and biochemically characterized) from the developing heart and reconstituted into a 3-dimensional culture system that closely approximates in situ morphological and biochemical sequences in ECT morphogenesis. Use is made of antisera and other binding proteins to test these hypotheses (a) in vitro (including tissue recombinations, membrane protein insertion, ECM derivatized styrene spheres etc.) and (b) in vivo by microinjection/microimplantation techniques. These experiments are designed to explain how macromolecules - relatively unstudied in early embryogenesis - could mechanistically regulate the development of endocardial cushion tissue.